1. Preparation of mismatched DNA substrates
All plasmids were propagated in TOP10 E. coli cells (Invitrogen) and isolated using a Qiagen Plasmid Maxi kit (Qiagen) followed by CsCl/ethidium bromide equilibrium centrifugation.
pSCW01 and pSCW02 were derived from pUC19CPDrev and follow the original numbering system with the four Nt.BstNBI endonuclease sites residing on the sense strand as defined by the pUC19 bla
gene in pSCW01 and the antisense strand in the case of pSCW02 () [5
]. A BbvC
1 restriction endonuclease site was introduced at position 55 (Stratagene QuikchangeXL) in pUC19CPDrev and an existing BbvC
1 site removed between positions 331-348. The resulting plasmid was digested with Aat
II and Sap
I, gel purified to remove the 76 bp intervening sequence, and ligated to synthetic 76 bp duplexes to create two 2030 bp plasmids, pSCW01 and pSCW02, that served as homoduplex DNA controls in MMR assays. The 76 bp sequence was created from two complementary oligonucleotides (Integrated DNA Technologies) that were heated at 85°C for 10 mins followed by slow cooling and then purification by native PAGE. Top strand: 5′- (p)CAT CGA GTC GGA TCC GAG TCA TTC CTG CAG CGA GTC CAT GGG AGT CAA ATA GAC AAC GAT TTG AAT TCG CTC TTC C -3′ (76mer) and bottom strand: 5′- (p)AGC GGA AGA GCG AAT TCA AAT CGT TGT CTA TTT GAC TCC CAT GGA CTC GCT GCA GGA ATG ACT CGG ATC CGA CTC GAT GAC GT -3′ (83mer) in the case of pSCW01 or top strand: 5′- (p)CAT CGA CTC GGA TCC GAC TCA TTC CTG CAG CGA CTC CAT GGG ACT CAA ATA GAC AAC GAT TTG AAT TCG CTC TTC C -3′ (76mer) and bottom strand: 5′- (p)AGC GGA AGA GCG AAT TCA AAT CGT TGT CTA TTT GAG TCC CAT GGA GTC GCT GCA GGA ATG AGT CGG ATC CGA GTC GAT GAC GT -3′ (83mer) in the case of pSCW02.
Figure 1 DNA mismatch repair substrates. (A) Map of essential features of pSCW01 and pSCW02 plasmids. Numbering is with respect to start of coding sequence of bla gene with sense strand on top. Red denotes nucleotide that is replaced to create a mismatch. Blue (more ...)
For the construction of mismatched substrates, 400 μg of pSCW01 or pSCW02 was incubated with 300 U of Nt.BstNBI endonuclease (New England BioLabs) at 55°C for 2 hr in 1.0 ml final volume in 1 × Buffer 3 (New England BioLabs) followed by the addition of another 300 U of Nt.BstNBI endonuclease and further incubation at 55°C for 2 hr. Nicking was monitored by gel electrophoresis. Displacement of the short, nicked fragments was facilitated by the addition of a 50-fold molar excess of displacer oligonucleotides (for pSCW01: 5′- ATT TGA CTC C-3′, 5′- CAT GGA CTC GCT GCA G-3′ and 5′-GAA TGA CTC GG-3′; for pSCW02: 5′-CAT CGA CTC GG-3′, 5′-ATC CGA CTC ATT CCT G-3′, and 5′-CAG CGA CTC C-3′) followed by 5 min at 85°C and then slow cooling to room temperature over 1.5 hr (). The resulting gapped DNA was precipitated in the presence of 13% (w/v) PEG 8000 and 10 mM MgCl2 by the addition of an equal volume of 26% (w/v) PEG 8000 and 20 mM MgCl2. Immediately after mixing, the sample was centrifuged at 12,000 × g at RT for 10 mins. The supernatants were discarded and the pellets were washed twice with 70% ethanol and resuspended in 50 μl of 10 mM Tris-HCl, pH 8.0. Gapped DNA was stored at -20°C.
A 10-fold molar excess of mismatched 37 nt oligonucleotides (For a G:T mismatch in pSCW01: 5′-(p)CCG AGT CAT TCC TGT AGC GAG TCC ATG GGA GTC AAA T-3′; for G:T in pSCW02: 5′-(p)GGA GTC GCT GCG GGA ATG AGT CGG ATC CGA GTC GAT G-3′; for meG:T in pSCW02: 5′-(p)GGA GTC GCT GCmeG GGA ATG AGT CGG ATC CGA GTC GAT G-3′) were annealed to the gapped pSCW01 or pSCW02 in 1 ml ligation buffer by incubation at 85°C for “G” oligonucleotide and 65°C for “O6meG” oligonucleotide for 10 min followed by slow cooling to room temperature. Ligation was monitored by gel electrophoresis after addition of 1 mM each DTT and ATP and incubation overnight at 16°C with 100 U of T4 DNA ligase (New England BioLabs). The mismatched DNA was extracted twice with phenol/chloroform and chloroform followed by ethanol precipitation. The DNA, resuspended in TE, was purified by CsCl/ethidium bromide equilibrium centrifugation followed by extraction in water-saturated n-butanol to remove ethidium bromide and dialysis against three 1-liter changes of TE buffer, pH 7.4. Covalently closed mismatched DNA was stored at -20°C. Nicked substrates were prepared by incubation with 20 U of Nt.BbvCI (5′ nick) or Nt.BspQI (3′ nick) (New England BioLabs) followed by phenol-chloroform extraction and ethanol precipitation (). The nicked mismatched substrates, pSCW01_GT, pSCW02_GT or pSCW02_mGT, were stored in TE, pH7.4, at -20°C.
2. Recombinant hMutSα and hMutLα
Recombinant hMutSα and hMutLα were produced in baculovirus-infected insect cells. Coding sequences for hMSH2 and hMSH6 were amplified by PCR from Image Clones (#3629489 and #4110354, respectively, Open Biosystem). The coding sequences for hMLH1 and hPMS2 were amplified by PCR from pFBdual_MutLα plasmid, a gift from Guo-Min Li at University of Kentucky Medical Center. Coding sequences for hMSH2, hMSH6, and hMLH1 were inserted into the pFastBac1 vector (Invitrogen) at BamHI and XhoI sites, and for hPMS2 at BamHI and SalI sites to yield pFB1_hMSH6, pFB1_hMSH2, pFB1_hMLH1, and pFB1_hPMS2. Constructs were verified by DNA sequencing and match Genbank accession BC004246 (hMSH6), BC021566 (hMSH2), U07343 (hMLH1) and U14658 (hPMS2). Baculovirus stocks were created (Kinnakeet Biotechnology) and propagated and titered in Sf9 cells (Invitrogen).
High Five insect cells (Invitrogen) were cultured in serum-free Express Five SFM medium (Invitrogen) supplemented with 20 mM L-glutamine at 27°C. Cells were coinfected at a density of 1×106/ml with baculovirus stocks encoding hMSH2 and hMSH6 at a multiplicity of infection (MOI) of 8 to produce MutSα, or with baculovirus constructs encoding hMLH1 and hPMS2 at a MOI of 2 to generate MutLα. One liter of infected cells was collected 60 hr later by centrifugation at 6,000 rpm for 10 min. For MutSα, cell pellets were dissolved in 80 ml of buffer A (25 mM HEPES, pH7.5; 0.1 mM EDTA; 10% glycerol; 1 mM DTT; 1 × Complete proteinase inhibitor cocktail (Roche); 0.1% PMSF). After swelling on ice for 10 min, cells were lysed with 20 strokes using a tight-fitting Dounce homogenizer on ice. After adjusting the salt concentration to 150 mM KCl, the suspension was clarified by centrifugation at 16,000 rpm for 30 min. The supernatant was then filtered through a 0.45μM filter unit (Corning) and loaded at 4°C onto a 6-ml Resource™ Q anion exchange column (GE Healthcare) equilibrated in Buffer A containing 150 mM KCl with a flow rate of 1.5 ml/min. The column was first washed with 60-ml buffer A plus 150 mM KCl, and then developed with a 90-ml gradient of KCl (150-650 mM) in buffer A. hMutSα eluted at approximately 250 mM KCl. The hMutSα-containing fractions were diluted to 150 mM KCl with buffer A and loaded onto a 5-ml HiTrap™ Heparin column (GE Healthcare) at 4°C which was developed with a 90-ml gradient of KCl, 150-650 mM, in buffer A; MutSα eluted at approximately 350 mM KCl. After concentration to 2 ml with an Amicon Ultracel-10K (Millipore), hMutSα-containing fractions were loaded on a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with buffer A containing 100 mM KCl. The pooled hMutSα fractions were aliquoted, frozen in liquid nitrogen and stored at -80 °C.
hMutLα was purified over Resource Q, Heparin and Superdex 200 columns as described for hMutSα with the following modifications. The lysis buffer contained 80 mM KCl; the 6-ml Resource™ Q anion exchange column and heparin column were equilibrated in buffer A containing 80 mM and 100 mM KCl, respectively. Columns were developed in a gradient of 100-450 mM KCl. hMutLα eluted at approximately 200 mM KCl on both Resource Q and heparin columns. The HiLoad 16/60 Superdex 200 column was equilibrated in buffer A containing 200 mM KCl. Concentrations of MutSα and MutLα were determined with a modified Bradford protein assay (Bio-Rad) using BSA as standard.
3. Mammalian cell culture and nuclear extracts
HeLaS3, MSH2-deficient LoVo, and MLH1-deficient HCT116 cells (American Type Culture Collection) were maintained at 37°C in DMEM (Invitrogen) with 10% fetal bovine serum (Invitrogen), 50 U penicillin, and 50 μg/ml streptomycin (Invitrogen) in a 5% CO2
, humidified atmosphere. Nuclear extracts were isolated following the previous studies but with some modifications [7
]. 4-5 × 108
cells were collected by centrifugation at 3000 × g for 5 min and washed once with 40 ml of cold hypotonic buffer (20 mM Hepes, pH 7.5; 5 mM KCl; 0.5 mM MgCl2
; 0.1% PMSF; 2 mM DTT; 1 μg/ml of each aprotinin, leupeptin, pefabloc, and E-64 (Roche)) containing 0.2 M sucrose. The cell pellet was resuspended in 10 ml of cold hypotonic buffer without sucrose, incubated on ice for 10 min, and then homogenized by 10 strokes of a loose-fitting Dounce homogenizer on ice. The resulting solution was centrifuged at 2000 × g for 5 min. The nuclear pellet was resuspended in 2.5 ml of extract buffer (50 mM Hepes, pH 7.5; 10% sucrose; 0.1% PMSF; 2 mM DTT; 1 μg/ml of each aprotinin, leupeptin, pefabloc, and E-64). After addition of 0.03 vol of 5 M NaCl, the nuclear suspension was mixed on a rotator for 1 hr at 4 °C and centrifuged at 15,000 × g for 30 min. The supernatant was concentrated to 5-8 mg/ml with an Amicon Ultracel-10K (Millipore), frozen in liquid nitrogen and stored at -80 °C.
4. Mismatch repair reactions (MMR)
In vitro MMR assays were performed with minor modification as previously described [5
] in a 40 μl volume containing 75 fmol (100 ng) of nicked pSCW01_GT DNA substrate, 100 μg of nuclear extract, 0.1 mM each of four dNTPs, in the standard MMR buffer condition of 20 mM Tris-HCl, pH 7.6; 1.5 mM ATP; 1 mM glutathione; 5 mM MgCl2
; 50 mg/ml BSA; the salt concentration was adjusted to 110 mM NaCl. After incubation at 37 °C for 15 min, the reaction was terminated by the addition of 80 μl of stop solution (25 mM EDTA, 0.67% sodium dodecyl sulfate, and 90 μg/ml proteinase K), then incubated at 37°C for another 15 min. DNA was extracted twice with an equal volume of phenol/chloroform and twice with chloroform. After precipitation with 2.5 volume of ethanol, DNA was dissolved in H2
O, digested with 4 units each of Pst
I and Ase
I endonuclease (New England Biolabs) and 1 μg of RNAase (Sigma) at 37°C for 1 hr, and then separated by electrophoresis on a 1.5% agarose gel. Alphaview 2.0 software (AlphaInnotek) was used to analyze the repair yield, which equals the ratio of the summed intensities of the 0.8- and 1.2-kb fragments to the total intensities of the 0.8-, the 1.2-kb, and the 2.0-kb bands. MMR assays for nicked pSCW02_GT substrate were performed as for pSCW01_GT. The recovered DNA was first cleaved with Fau
I (New England Biolabs) plus RNAase at 55°C for 1 hr, then digested with Ase
I at 37°C for another 1 hr. Repair products yield 0.8- and 1.2-kb fragments.
5. Analysis of excision gaps
To measure the extent of excision generated during MMR, we omitted exogenous dNTPs from a standard MMR reaction mixture containing nicked mismatched DNA substrate or various control substrates, nuclear extract and 20-40 nM hMutSα and 20-40 nM hMutLα as indicated. Incubation was at 37 °C for 7 min. The reaction was terminated by stop solution. DNA was extracted with phenol/chloroform and precipitated with ethanol as described above. Excision was measured in one of three ways employing adaptations of previously described excision assays [5
5.1 Restriction endonuclease cleavage
The gapped DNA was digested with BamHI and AseI (New England Biolabs) and RNAase at 37 °C for 1 hr and then separated by electrophoresis on a 1.5% agarose gel. Since the BamHI endonuclease cleavage site is located between the mismatch site and the 5′ nick site, excision initiating at the nick and extending beyond the mismatch will result in loss of the BamHI site resulting in a 2 kb linearized DNA.
5.2 Annealing to oligonucleotide probes
The recovered DNA was digested with AseI plus RNAase, annealed with 0.2 pmol of a particular 32P-labeled oligomer probe (5′-GTC ATT CCT GCA GCG AGT CCA TGG GAG TCA-3′), and then separated from free oligomers by electrophoresis. DNA bands were measured quantitatively by ethidium bromide staining to normalize DNA recovery and loading. The gels were dried and the radioactivity was measured with a Fuji phosphoimager BSA-2500.
5.3 Southern blot analysis
The excision products were digested with AflII (New England BioLabs) plus RNAase and mixed with 2 × formamide loading buffer (95% formamide, 10 mM NaOH, 0.25% Bromophenol blue, 0.25% Xylene cyanol). The mixture was heated at 85°C for 5 min and cooled on ice followed by electrophoresis on 6% denaturing polyacrylamide gels and transfer to Hybond-NX membranes (Amersham). Membranes were cross-linked with a UV crosslinker (Stratagene), and then pre-hybridized at 37 °C for 1 hr with hybridization solution (0.2% SDS, 0.5% PVP, 0.2% Heparin, 0.05 M Tris pH 7.5, 1 M NaCl, 1 mM EDTA). After incubation with a 32P-5′ end-labeled oligonucleotide probe (5′-TTG GAG CGA ACG ACC TAC ACC GA-3′) overnight at 37°C, the membranes were washed twice with washing solution I (2 × SSC, 0.1% SDS) at 37°C, and twice with washing solution II (1 × SSC, 0.1% SDS). Membranes containing reaction products were analyzed using a Fuji phosphoimager BAS-2500 and ImageGauge V4.22.